1. Field of the Invention
The present disclosure relates to primers and methods for detecting variant(s). More particularly, the present disclosure relates to a self-competitive primer for preferentially amplifying a sample nucleic acid having a nucleotide variation.
2. Description of Related Art
Gene mutations are alterations in the nucleotide sequence of a given gene or regulatory sequence from a naturally occurring or normal nucleotide sequence. A mutation may be a point mutation (single nucleotide substitution), a deletion or insertion mutation of one or more nucleotides, a substitution mutation of more than one nucleotide, or crossing-over in chromosomal level (translocation).
The identification of gene mutations or nucleotide variations is of great importance in molecular genetics. For example, many genes associated with the onset and/or progression of cancers have been identified, and thereby allows the development of molecular target therapies that specifically target these genes. In these target therapies, sequence variation of the targeted gene(s) may affect the efficacy of a given treatment. Therefore, the detection of gene mutation(s) in cancerous cells makes it possible to devise a treatment plan best suited the patient.
Various techniques for detecting gene mutations or nucleotide variations have been developed. For examples, mutated nucleotide(s) could be revealed by Sanger's direct sequencing of sequence of interest. However, the sensitivity of this technique is too low and the operation thereof requires considerable time and effort, thereby hindering its application in clinical and research uses.
Alternatively, primers and/or probes specific to the nucleotide variation could be used to positively detect such variation. Conventionally, primers or probes for detecting mutations are designed with prior knowledge of the sequence of the mutated site. For example, allele specific oligonucleotides-polymerase chain reaction (ASO-PCR) is often used for the detection of gene mutations. In ASO-PCR, a primer specific to the wild-type or the mutant sequence is used, and the presence or absence of a mutant sequence is judged by whether amplification may proceed. One disadvantage of ASO-PCR lies in that it generally has a low specificity thereby leading to a high false-positive rate. Moreover, the low specificity may result in mis-amplification of the sample nucleic acid, thereby producing amplicons having a sequence differing from the sample nucleic acid by introducing the primer sequence into the amplicons. The mis-amplification of the sample nucleic acid would render the subsequent confirmation step (such as sequencing) meaningless since the amplicon does not reflect the amplification of the genuine sequence of the sample nucleic acid. Further, in the ASO-PCR method, only one primer is allowed in one reaction system, and therefore, it is only possible to detect one sequence (either wild-type or mutant) once. Therefore, in the event where multiple nucleotide variations may occur at a same position, multiple primers shall be designed to ensure the full coverage of all mutated sequences, and separate reactions shall be conducted in order to correctively detect the mutation.
Another commonly used detection technique is ligase chain reaction (LCR), which is often utilized in conjunction with other amplification-based methods such as PCR. LCR employs a thermostable ligase and two sets of primers in which each primer set has two primers that are ligated together only when immediately adjacent to each other thereby allowing the discrimination of single nucleotide variation (such as point mutation, single-nucleotide deletion, and single-nucleotide insertion). Despite having various advantages, LCR is not capable of detecting a mutated sequence that has multiple-nucleotide variation.
Moreover, in the event of disease diagnosis, samples obtained from living tissue may contain cells having the wild-type sequence, as well as the mutated sequence. As could be appreciated, the wild-type cells often present in an excess amount with respect to mutated cells, which may obscure the amplification and thus the detection of the mutated sequence.
In view of the foregoing, there exists a need in the art for the development of a detection method capable of accurately detecting nucleotide variation, including both single-nucleotide and multiple-nucleotide variations.